U.S. patent application number 12/370581 was filed with the patent office on 2009-08-27 for apparatus and method for obtaining images using coherent anti-stokes raman scattering.
This patent application is currently assigned to Gwangju Institute of Science and Technology. Invention is credited to Seung Bum CHO, Dug Young KIM.
Application Number | 20090213370 12/370581 |
Document ID | / |
Family ID | 40589988 |
Filed Date | 2009-08-27 |
United States Patent
Application |
20090213370 |
Kind Code |
A1 |
KIM; Dug Young ; et
al. |
August 27, 2009 |
APPARATUS AND METHOD FOR OBTAINING IMAGES USING COHERENT
ANTI-STOKES RAMAN SCATTERING
Abstract
Disclosed is an apparatus and method for obtaining images using
coherent anti-stokes Raman scattering. The apparatus for obtaining
images using coherent anti-stokes Raman scattering according to the
present invention comprises: a pump light source and a stokes light
source that irradiate pump light and stokes light on a sample to
generate anti-stokes light having anti-stokes frequency; a
reference light source that generates reference light; and an image
obtaining unit that obtains the images of the sample using a change
in phase of the reference light due to a change in the refractive
index of the sample in the vicinity of the anti-stokes frequency.
Thereby, the present invention can provide the apparatus for
obtaining images using coherent anti-stokes Raman scattering that
is not affected by a non-resonant background signal phenomenon,
strong resistance against noise even in a weak signal, and has
excellent sensitivity and resolution.
Inventors: |
KIM; Dug Young; (Gwangju,
KR) ; CHO; Seung Bum; (Gwangju, KR) |
Correspondence
Address: |
AMPACC LAW GROUP
3500 188th St. SW
Lynnwood
WA
98037
US
|
Assignee: |
Gwangju Institute of Science and
Technology
Gwangju
KR
|
Family ID: |
40589988 |
Appl. No.: |
12/370581 |
Filed: |
February 12, 2009 |
Current U.S.
Class: |
356/301 |
Current CPC
Class: |
G01N 2021/653 20130101;
G01J 3/44 20130101; G01N 21/65 20130101 |
Class at
Publication: |
356/301 |
International
Class: |
G01J 3/44 20060101
G01J003/44 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 27, 2008 |
KR |
10-2008-0017799 |
Claims
1. An apparatus for obtaining images using coherent anti-stokes
Raman scattering comprising: a pump light source and a stokes light
source that irradiate pump light and stokes light on a sample to
generate anti-stokes light having anti-stokes frequency; a
reference light source that generates reference light; and an image
obtaining unit that obtains the images of the sample using a change
in phase of the reference light due to a change in the refractive
index of the sample in the vicinity of the anti-stokes
frequency.
2. The apparatus for obtaining images according to claim 1, wherein
the reference light has higher or lower frequency by a
predetermined magnitude than the anti-stokes frequency.
3. The apparatus for obtaining images according to claim 1, wherein
the image obtaining unit includes a unit that splits a path to a
first path passing through the sample and a second path not passing
through the sample and propagates a reference light to the first
and second paths and obtains the images of the sample using an
interference phenomenon due to a phase difference between the
reference light passing through the first path and the reference
light passing through the second path.
4. The apparatus for obtaining images according to claim 1, wherein
the reference light source includes a first reference light source
and a second light source each generating first reference light
having higher frequency by predetermined magnitude than the
anti-stokes frequency and second reference light having lower
frequency by predetermined magnitude than the anti-stokes
frequency, and the image obtaining unit includes a unit that splits
a path into the first path passing through the sample and the
second path not passing through the sample and propagates each of
the first and second reference lights to the first and second paths
and obtains the images of the sample using a phase difference
between the first reference light passing through the first path
and the first reference light passing through the second path and a
phase difference between the second reference light passing through
the first path and the second reference light passing through the
second path together.
5. The apparatus for obtaining images according to claim 1, wherein
the stokes light source generates stokes light having a broad
frequency band to generate anti-stokes light having a plurality of
anti-stokes frequencies, the reference light source generates
reference light having a broad frequency band, and the image
obtaining unit obtains the images of the sample using a change in
phase of the reference light due to a change in the refractive
index of the sample in the vicinity of each of the plurality of
anti-strokes frequencies.
6. An apparatus for obtaining images using coherent anti-stokes
Raman scattering comprising: a pump light source and a stokes light
source that irradiate pump light and stokes light on a sample to
generate anti-stokes light having anti-stokes frequency; a
reference light source that generates reference light; and an image
obtaining unit that obtains the images of the sample using a change
in birefringence of the sample in the vicinity of the anti-stokes
frequency.
7. The apparatus for obtaining images according to claim 6, wherein
the image obtaining unit includes a first polarizer that is
installed at a position before the reference light propagated to
paths passes through the sample and a second polarizer that is
installed at a position before the reference light passes through
the sample and has an optical axis in a direction different from
the first polarizer.
8. The apparatus for obtaining images according to claim 6, wherein
the image obtaining unit further includes an electromagnetic field
generator that applies electric field or magnetic field to the
sample.
9. A method for obtaining images using coherent anti-stokes Raman
scattering comprising: (a) irradiating pump light and stokes light
on a sample to generate anti-stokes light having anti-stokes
frequency; (b) generating reference light; and (c) obtaining the
images of the sample using a change in phase of the reference light
due to a change in refractive index of the sample in the vicinity
of the anti-stokes frequency.
10. The method for obtaining images according to claim 9, wherein
the reference light has higher or lower frequency by a
predetermined magnitude than the anti-stokes frequency.
11. The method for obtaining images according to claim 9, wherein
step (c) include: c1) splitting a path into a first path passing
through the sample and a second path not passing through the sample
and propagating the reference light to the first and second paths;
and c2) obtaining the images of the sample using an interference
phenomenon due to a phase difference between the reference light
passing through the first path and the reference light passing
through the second pa th.
12. The method for obtaining images according to claim 9, wherein
step (b) includes generating first reference light having higher
frequency by a predetermined magnitude than the anti-stokes
frequency and second reference light having lower frequency by a
predetermined magnitude than the anti-stokes frequency, and step
(c) includes, c1) splitting a path into the first path passing
through the sample and the second path not passing through the
sample and propagating each of the first and second reference
lights to the first and second paths; and c2) obtaining the images
of the sample using a phase difference between the first reference
light passing through the first path and the first reference light
passing through the second path and a phase difference between the
second reference light passing through the first path and the
second reference light passing through the second path
together.
13. The method for obtaining images according to claim 9, wherein
step (a) irradiates stokes light having a broad frequency band on
the sample to generate anti-stokes light having a plurality of
anti-stokes frequencies, step (b) generates the reference light
having a broad frequency band, and step (c) obtains the images of
the sample using a change in phase of the reference light due to a
change in refractive index of the sample in the vicinity of each of
the plurality of anti-strokes frequencies.
14. A method for obtaining images using coherent anti-stokes Raman
scattering comprising: (a) irradiating pump light and stokes light
on a sample to generate anti-stokes light having anti-stokes
frequency; (b) generating reference light; and (c) obtaining the
images of the sample using a change in birefringence of the sample
in the vicinity of the anti-stokes frequency.
15. The method for obtaining images according to claim 14, wherein
step (c) includes, (c1) analyzing polarizing property before the
reference light passes through the sample and polarizing property
after the reference light passes through the sample and measuring
birefrigence of the sample, and (c2) imaging the measured
results.
16. The method for obtaining images according to claim 14, further
comprising electric field or magnetic field to the sample.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Technical Field
[0002] The present invention relates to an apparatus and method for
obtaining images to reveal structures and properties of materials
and more particularity, to an apparatus and method for obtaining
images using coherent anti-stokes Raman scattering.
[0003] 2. Related Art
[0004] Raman spectroscopy is a phenomenon that scatters light
having a wavelength different from that of monochromatic light
input from a sample on which the monochromatic light is irradiated.
Recently, the Raman spectroscopy tracks a change of a vibration
mode together with infrared spectroscopy, such that it has
established an independent area of learning that reveals structures
and properties of molecules.
[0005] Even though the Raman spectroscopy was studied earlier than
the infrared spectroscopy due to characteristic using Raman
scattering light having weak strength, it has been slower in
development. As a result, the frequency in the use of the Raman
spectroscopy has been low until now. However, with the advent of a
laser having improved output strength, the Raman spectroscopy is
being developed rapidly. As a result, the use of the Raman
spectroscopy is currently of interest for various applications.
[0006] Meanwhile, a fluorescent microscope is considered as the
epochal technology in the field of cell biology due to the help of
the development of various fluorescence probes, confocal detection,
and three-dimensional imaging through multi-photon excitation. The
fluorescent microscope irradiates a light source at a wavelength
that can excite fluorescent molecules, which is known as the
fluorescence probes naturally existing in the sample or
artificially injected into the sample, on the sample. At this time,
the sample emits fluorescence by absorbing the excitation light
source and is observed using a filter that selectively transmits
fluorescence. The fluorescent microscope is advantageous in that it
has higher resolution than a general optical microscope. However,
since the fluorescent microscope uses the fluorescence probes, it
has problems in that it changes the sample to be measured and
causes a photobleaching phenomenon.
[0007] A microscope using coherent anti-stokes Raman scattering
(CARS) that inputs fixed/variable laser lights to Raman active
media and measures spectrums of anti-stokes light obtained by a
combination thereof has been widely used on the grounds that it has
high sensitivity and is not affected by the media generating
fluorescence.
[0008] FIG. 1 is an energy band diagram for explaining a generation
principle of a general CARS signal. The CARS is a four wave mixing
process that generates anti-stokes light by interacting pump light,
stokes light, and probe light with a sample and uses two laser
beams having different frequencies. A first laser beam serves as
the stokes light (.omega..sub.s) and a second laser beam serves as
the pump light (.omega..sub.p) and the probe light
(.omega..sub.pro). In other words, the frequency of .omega..sub.pro
is identical with .omega..sub.p. The electric field of the light is
EP (.omega..sub.p), ES (.omega..sub.s) , Epro (.omega..sub.pro)
(=EP (.omega..sub.p)). If the laser beam is irradiated, electrons,
which are in a ground state .nu..sub.0=0 are first excited in a
virtual state by the pump light (.omega..sub.p) and most of the
electrons are then transitioned into a level .nu..sub.1=1 by the
stokes light .omega..sub.s. At this time, the inherent vibration
frequency of the electrons becomes .OMEGA. by the resonant Raman
scattering. After the electrons transitioned from .nu..sub.0=0 to
.nu..sub.0=1 are back excited into a virtual state such as
.omega..sub.p+.OMEGA. by the probe light (.omega..sub.pro), they
emit the anti-stokes light having frequency
.omega..sub.as=2.omega..sub.p-.omega..sub.s that satisfies energy
conservation, that is, CARS signals, and are transitioned to the
level .nu..sub.0=0. The CARS microscope analyzes materials by
measuring the strength of the CARS signal.
[0009] Since the CARS microscope as described above has the same
resolution as the confocal microscope but does not use emission of
a pigment, it does not change the sample. Further, since the CARS
microscope uses Raman that corresponds to a vibration level of
chemical species, it has an advantage in that it can select the
chemical species. Moreover, the CARS microscope can obtain a very
large signal as compared to spontaneous Raman scattering and
because it has anti-stokes frequency different from the frequencies
used by the two laser beams, the signal can be easily separated by
using a filter, etc.
[0010] However, a representative disadvantage of the coherent
anti-stokes Raman scattering is a non-resonant background signal
phenomenon caused due to two-photon electronic resonance, which has
been studied by [M. D. Duncan, J. Reintjes, and T. J. Manuccia,
"Scanning coherent anti-Stokes Raman microscope," Opt. Lett. 7,
350-352, 1982]. It was found that any two-photon-enhanced
background phenomenon exceeding the resonant vibration signal is
generated due to the use of visible light. In a study by [A.
Zumbusch, G. R. Holtom, and X. S. Xie, "Three-dimensional
vibrational imaging by coherent anti-Stokes Raman scattering,"
Phys. Rev. Lett. 82, 4142-4145, 1999.], it was found that
two-photon electronic resonance is prevented and sensitivity
increase, by using near-infrared light. Thereafter, with the
development of the coherent anti-stokes Raman scattering
spectrometer, several methods were used for reducing the
non-resonant background signal.
[0011] However, even though there are many problems in the
conventional technologies associated with the CARS, these
technologies all detected the strength of the CARS signal and
directly analyzed them. As a result, these technologies focused on
the difference between the CARS signal and the non-resonant
background signal, such that many problems occurred. In particular,
a method focusing on the reduction of the non-resonant background
signal weakens the strength of light to be detected, such that
there is a need for a high-specification photo detector in order to
detect the signal having weak strength without noise. Further,
since the method directly analyzes the signal to be detected by the
strength of signal, it has limitations in sensitivity, resolution,
accuracy, etc.
SUMMARY OF THE INVENTION
[0012] It is an object of the present invention to provide an
apparatus for obtaining images using coherent anti-stokes Raman
scattering, which is not affected by a non-resonant background
signal phenomenon, has strong resistance against noise even in a
weak signal, and has excellent sensitivity and resolution.
[0013] It is another object of the present invention to a method
for obtaining images using coherent anti-stokes Raman scattering,
which is not affected by a non-resonant background signal
phenomenon, has strong resistance against noise even in a weak
signal, and has excellent sensitivity and resolution.
[0014] In order to achieve the above object, there is provided an
apparatus for obtaining images using coherent anti-stokes Raman
scattering according to the present invention, comprising: a pump
light source and a stokes light source that irradiate pump light
and stokes light on a sample to generate anti-stokes light having
anti-stokes frequency; a reference light source that generates
reference light; and an image obtaining unit that obtains the
images of the sample using a change in phase of the reference light
due to a change in the refractive index of the sample in the
vicinity of the anti-stokes frequency.
[0015] Preferably, the reference light has higher or lower
frequency by a predetermined magnitude than the anti-stokes
frequency.
[0016] Further, the image obtaining unit includes a unit that
splits a path to a first path passing through the sample and a
second path not passing through the sample and propagates a
reference light to the first and second paths and may obtain the
images of the sample using an interference phenomenon due to a
phase difference between the reference light passing through the
first path and the reference light passing through the second
path.
[0017] Also, the reference light source includes a first reference
light source and a second light source each generating first
reference light having higher frequency by predetermined magnitude
than the anti-stokes frequency and second reference light having
lower frequency by a predetermined magnitude than the anti-stokes
frequency, and the image obtaining unit includes a unit that splits
a path into the first path passing through the sample and the
second path not passing through the sample and propagates each of
the first and second reference lights to the first and second paths
and may obtain the images of the sample using a phase difference
between the first reference light passing through the first path
and the first reference light passing through the second path and a
phase difference between the second reference light passing through
the first path and the second reference light passing through the
second path together.
[0018] In addition, the stokes light source generates stokes light
having a broad frequency band to generate anti-stokes light having
a plurality of anti-stokes frequencies, the reference light source
generates reference light having a broad frequency band, the image
obtaining unit may obtain the images of the sample using a change
in phase of the reference light due to the change in refractive
index of the sample in the vicinity of each of the plurality of
anti-strokes frequencies.
[0019] In order to achieve the above object, there is provided an
apparatus for obtaining images using coherent anti-stokes Raman
scattering according to the present invention, comprising: a pump
light source and a stokes light source that irradiate pump light
and stokes light on a sample to generate anti-stokes light having
anti-stokes frequency; a reference light source that generates
reference light; and an image obtaining unit that obtains the
images of the sample using the change in birefringence of the
sample in the vicinity of the anti-stokes frequency.
[0020] The image obtaining unit may include a first polarizer that
is installed at a position before the reference light propagated to
paths passes through the sample and a second polarizer that is
installed at a position before the reference light passes through
the sample and has an optical axis in a direction different from
the first polarizer.
[0021] Further, the image obtaining unit may further include an
electromagnetic field generator that applies electric field or
magnetic field to the sample.
[0022] In order to achieve another object, there is provided a
method for obtaining images using coherent anti-stokes Raman
scattering according to the present invention, comprising: (a)
irradiating pump light and stokes light on a sample to generate
anti-stokes light having anti-stokes frequency; (b) generating
reference light; and (c) obtaining the images of the sample using a
change in phase of the reference light due to a change in
refractive index of the sample in the vicinity of the anti-stokes
frequency.
[0023] Step (c) may include cl) splitting a path into a first path
passing through the sample and a second path not passing through
the sample and propagating the reference light to the first and
second paths, and c2) obtaining the images of the sample using an
interference phenomenon due to a phase difference between the
reference light passing through the first path and the reference
light passing through the second path.
[0024] Also, step (b) may include generating first reference light
having higher frequency by predetermined magnitude than the
anti-stokes frequency and second reference light having lower
frequency by predetermined magnitude than the anti-stokes
frequency, and step (c) includes c1) splitting a path into the
first path passing through the sample and the second path not
passing through the sample and propagating each of the first and
second reference lights to the first and second paths and c2)
obtaining the images of the sample using a phase difference between
the first reference light passing through the first path and the
first reference light passing through the second path and a phase
difference between the second reference light passing through the
first path and the second reference light passing through the
second path together.
[0025] Further, step (a) irradiates stokes light having a broad
frequency band on the sample to generate anti-stokes light having a
plurality of anti-stokes frequencies, step (b) generates the
reference light having a broad frequency band, and step (c) may
obtain the images of the sample using the change in phase of the
reference light due to a change in refractive index of the sample
in the vicinity of each of the plurality of anti-strokes
frequencies.
[0026] In order to solve another object, there is provided a method
for obtaining images using coherent anti-stokes Raman scattering
according to the present invention, comprising: (a) irradiating
pump light and stokes light on a sample to generate anti-stokes
light having anti-stokes frequency; (b) generating reference light;
and (c) obtaining the images of the sample using a change in
birefringence of the sample in the vicinity of the anti-stokes
frequency.
[0027] Step (c) includes (c1) analyzing polarizing property before
the reference light passes through the sample and polarizing
property after the reference light passes through the sample and
measuring birefrigence of the sample, and (c2) imaging the measured
results.
[0028] Moreover, the a method for obtaining images using coherent
anti-stokes Raman scattering may further include applying electric
field or magnetic field to the sample.
[0029] The present invention can provide the apparatus and method
for obtaining images using coherent anti-stokes Raman scattering,
which is not affected by a non-resonant background signal
phenomenon, has strong resistance against noise even in a weak
signal, and has excellent sensitivity and resolution.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1 is an energy band diagram for explaining a generation
principle of a general CARS signal;
[0031] FIG. 2 is a graph for explaining the appearance of how the
refractive index is changed in the vicinity of anti-stokes
frequency due to coherent anti-stokes Raman scattering;
[0032] FIG. 3 is a graph showing in more detail a change in
absorptance and refractive index;
[0033] FIG. 4 is a diagram showing a configuration of an apparatus
for obtaining images using the coherent anti-stokes Raman
scattering according to one embodiment of the present
invention;
[0034] FIG. 5 is a graph showing appearance, absorptance, and
refractive index of anti-stokes light generated when a frequency
band of stokes light is broad;
[0035] FIG. 6 is a graph for explaining an embodiment using two
reference lights together in another embodiment of the present
invention;
[0036] FIG. 7 is a diagram showing a configuration of an apparatus
for obtaining images using coherent anti-stokes Raman scattering
according to another embodiment of the present invention;
[0037] FIG. 8 is a diagram showing a configuration of an apparatus
for obtaining images using coherent anti-stokes Raman scattering
according to still another embodiment of the present invention;
and
[0038] FIG. 9 is a diagram showing a configuration of an apparatus
for obtaining images using coherent anti-stokes Raman scattering
according to still another embodiment of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] Hereinafter, preferred embodiments of the present invention
will be described in detail with reference to the accompanying
drawings. First of all, we should note that in giving reference
numerals to elements of each drawing, like reference numerals refer
to like elements even though like elements are shown in different
drawings. Further, in describing the present invention, well-known
functions or constructions will not be described in detail since
they may unnecessarily obscure the understanding of the present
invention.
[0040] The present invention implements an apparatus and method for
obtaining images that is not affected by a non-resonant background
signal phenomenon and has strong resistance against noise even if
signals are weak without measuring signal strength of anti-stokes
light generated by coherent anti-stokes Raman scattering but
indirectly using it. To this end, the apparatus and method for
obtaining images according to the present invention uses a
principle where refractive index of a sample is changed in the
vicinity of anti-stroke frequency having the anti-stroke light
generated by the coherent anti-stroke Raman scattering.
[0041] FIG. 2 is a graph for explaining the appearance of how the
refractive index is changed in the vicinity of anti-stokes
frequency due to coherent anti-stokes Raman scattering. Referring
to FIG. 2, .omega..sub.p represents frequency of pump light and
.omega..sub.s represents frequency of stokes light. If the pump
light and the stokes light are irradiated on a sample, the
anti-stokes light having .omega..sub.as(=2(p-(s) is emitted from
the sample. At this time, absorptance or emission of light is
generated in the vicinity of the anti-stokes frequency, such that
the absorptance ((or emission rate) is changed. Absorptance n(( )
of light according to frequency in the vicinity of (s is shown in
FIG. 2. The change in absorptance accompanies the change in
refractive index. The change in absorptance and the change in
absorptance depend on the following equation that is referred to as
Kramer-Kronig relation.
.DELTA. n ( .omega. ' ) = c .pi. P .intg. 0 .infin. .DELTA. a (
.omega. ) .omega. .omega. 2 - .omega. '2 [ Equation 1 ]
##EQU00001##
[0042] where c is any constant and P means Cauchy principal
value.
[0043] Refractive index n (.omega.)) according to the relation
between the absorptance and the refractive index is shown in FIG.
2. As shown in FIG. 2, the refractive index is changed most in the
vicinity of .omega..sub.as.
[0044] FIG. 3 is a graph showing in more detail a change in
absorptance and refractive index. Referring to FIG. 3, a refractive
index graph n.sub.1 corresponding to an absorptance graph
.alpha..sub.2 is shown in FIG. 3. As shown in FIG. 3, the most
change in refractive index is at lower or higher frequency by a
predetermined frequency in the vicinity of .omega..sub.as. Also, it
can be appreciated that as the change in absorptance is large based
on .omega..sub.as, the change in refractive index becomes larger in
the vicinity of .omega..sub.as.
[0045] FIG. 4 is a diagram showing a configuration of an apparatus
for obtaining images using the coherent anti-stokes Raman
scattering according to one embodiment of the present invention.
Referring to FIG. 4, the apparatus for obtaining images according
to the embodiment includes a pump light source 401 and a stokes
light source 402 that generate pump light and stokes light
irradiated on the sample to generate anti-stokes light having
anti-stokes frequency, a reference light source 410 that generates
reference light, and as shown in FIG. 4, an image obtaining unit
that obtains images of the sample using the change in phase of the
reference light due to the change in refractive index of the sample
in the vicinity of the anti-stroke frequency. As the pump light
source 401 and the stokes light source 402, a pulse type laser
light source is used.
[0046] With the embodiment, the frequency .omega..sub.ref of the
reference light has higher or lower frequency by a predetermined
magnitude than the anti-stokes frequency .omega..sub.as. At this
time, it is preferable to have frequency corresponding to a point
where the change in the refractive index is largest. For example,
as shown in FIGS. 1 and 2, the frequency corresponding to the point
where the change in refractive index is largest, that is, lower
frequency by the predetermined magnitude than .omega..sub.as as the
frequency of the reference light .omega..sub.ref. Of course, it may
use the higher frequency by the predetermined magnitude than
.omega..sub.as as the frequency of the reference light.
[0047] Hereinafter, an operation of the apparatus for obtaining
images according to the embodiment shown in FIG. 4 will be
described. The pump light source 401 and the stokes light source
402 each generates the pump light at the frequency .omega..sub.p
and the stokes light at the frequency .omega..sub.s and the pump
light and the stokes light are reflected from a first dichroic
reflector 413 and are irradiated on the sample S. In the sample,
the anti-stokes light is generated due to the coherent anti-stokes
Raman scattering. And, the pump light and the stokes light are
reflected from a second dichroic reflector 415 and emitted to the
outside. The first dichroic reflector 413 and the second dichroic
reflector 415 perform a function of filtering the pump light and
the stokes light by reflecting the pump light at the frequency
.omega..sub.p and the stokes light at the frequency .omega..sub.s
and passing through the reference light at the frequency
.omega..sub.ref.
[0048] The reference light source 410 generates the reference light
having the frequency .omega..sub.ref. The reference light is split
into a first path passing through the sample S and a second path
not passing through the sample S by a first optical splitter 411.
At this time, the reference light split into the first path is
reflected from the first reflector 412 and passes through the first
dichroic reflector 413 and then passes through the sample S. At
this time, the reference light can be magnified by a microscope
object lens 414. As described above, in the sample S, since the
change in the refractive index occurs in the vicinity of the
anti-stokes frequency due to the coherent anti-stokes scattering,
the reference light passing through the sample is subjected to the
phase change. This reference light is incident to a second optical
splitter 418.
[0049] The reference light split into the second path is reflected
from the second reflector 416 and is incident to the second optical
splitter 418. At this time, in order to maximize the interference
pattern, a variable wavelength plate 417 may be disposed at the
path of the reference light.
[0050] There is a phase difference between the reference light
propagated to the first path passing through the sample and the
reference light propagated to the second path not passing through
the sample. Therefore, the interference phenomenon occurs after the
reference light passes through the second optical splitter 418. The
detector 419 detects signals due to the interference phenomenon and
the detected signal is transferred to an imaging unit 430. As the
detector 419, for example, a charge coupled device can be used. The
imaging unit 430 analyzes the detected signal, images it, and
outputs it through a display (not shown), etc.
[0051] In the above-mentioned embodiment, in order to change a
relative length of the path between the reference light propagated
to the first path and the reference light propagated to the second
path, each component, for example, a transfer device, which
transfers the first optical splitter 411, the first and second
reflectors 412 and 416, and the like, may be installed.
[0052] Further, as a modified embodiment of the above-mentioned
embodiment, a pulse type laser light source as the pump light
source 401 is used and a broadband light source having a broad
frequency band instead of a single frequency of a pulse type laser
light source as the stokes light source 402, for example, an LED
light source is used.
[0053] FIG. 5 is a graph showing appearance, absorptance, and
refractive index of anti-stokes light generated when a frequency
band of stokes light is broad. As shown in FIG. 5, when the
anti-stokes light has a sufficient broad frequency band, the
coherent anti-stokes Raman scattering is generated at a plurality
of frequencies to generate the anti-stokes light having a plurality
of anti-stokes frequencies .omega..sub.asi and .omega..sub.as2.
Therefore, the images of the sample can be obtained using the
change in phase of the reference light due to the change in the
refractive index of the sample at each frequency. To this end, in
the present embodiment, the reference light also uses the reference
light having the broad frequency band. As the detector 419, a
device capable of detecting the signals generated due to the
interference phenomenon at several frequency bands is used. For
example, an arrayed detector or a CCD array, etc., may be used.
[0054] FIG. 6 is a graph for explaining an embodiment using two
reference lights, that is, two reference lights each having a lower
frequency .omega..sub.ref1 by the predetermined magnitude than the
anti-stokes frequency .omega..sub.as and higher frequency
.omega..sub.ref2 by the predetermined magnitude than the
anti-stokes frequency .omega..sub.as together as another embodiment
of the present invention. As shown in FIG. 6, the sign of the
refractive index at .omega..sub.ref1 and the refractive index at
.omega..sub.ref2 is opposite to each other based on the refractive
index at the anti-stokes frequency .omega..sub.as. Therefore, the
noise at the time of measuring the change in strength of the light
source, etc. can be reduced by using the difference between the
refractive indexes at two frequencies.
[0055] FIG. 7 is a diagram showing a configuration of an apparatus
for obtaining images using coherent anti-stokes Raman scattering
according to another embodiment of the present invention and is a
configuration diagram showing an embodiment using two reference
lights each having two frequencies of .omega..sub.ref1 and
.omega..sub.ref2.
[0056] Referring to FIG. 7, an apparatus for obtaining images
according to the present embodiment includes a pump light source
701 and a stokes light source 702 that generate pump light and
stokes light irradiated on the sample to generate anti-stokes light
having anti-stokes frequency, a first reference light source 703
and a second reference light source 704 that generate first
reference light at .omega..sub.ref1 and second reference light at
.omega..sub.ref2, a unit that splits a path into the first path
passing through the sample S and the second path not passing
through the sample S and propagates each of the first and second
reference lights to the first and second paths and an image
obtaining unit that obtain the images of the sample using a phase
difference between the first reference light passing through the
first path and the first reference light passing through the second
path and a phase difference between the second reference light
passing through the first path and the second reference light
passing through the second path together.
[0057] Hereinafter, the operation of the apparatus for obtaining
images according to the embodiment shown in FIG. 7 will be
described. The pump light source 701 and the stokes light source
702 each generates the pump light at the frequency .omega..sub.p
and the stokes light at the frequency .omega..sub.s and the pump
light and the stokes light are reflected from a second dichroic
reflector 713 and are irradiated on the sample S. In the sample S,
the anti-stroke light is generated due to the coherent anti-stokes
Raman scattering. The pump light and the stokes light are reflected
from a third dichroic reflector 715 and are discharged to the
outside. The second dichroic reflector 713 and the third dichroic
reflector 715 reflect the pump light at the frequency .omega..sub.p
and the stokes light at the frequency .omega..sub.s while
performing as a filter for the pump light and the stokes light by
passing through the pump light at the frequency .omega..sub.ref1
and the stokes light at the frequency .omega..sub.ref2.
[0058] The first reference light source 703 generates the first
reference light having the frequency .omega..sub.ref1. The first
reference light is reflected from the first reflector 705 and the
first dichroic reflector 706 and is incident on the first splitter
711, which is in turn split into two paths, where the first path
passes through the sample S and the second path does not pass
through the sample S.
[0059] The second reference light source 704 generates the second
reference light having the frequency .omega..sub.ref2. The second
reference light passes through the first dichroic reflector 706 and
is incident on the first splitter 711, which is in turn split into
two paths, where the first path passes through the sample S and the
second path does not pass through the sample S like the first
reference light.
[0060] The first reference light and the second reference light
split into the first path are reflected from the second reflector
712 and passes through the sample S via the second dichroic
reflector 713. At this time, the reference light can be magnified
by a microscope object lens 714. As described above, in the sample
S, since the change in refractive index occurs in the vicinity of
the anti-stokes frequency due to the coherent anti-stokes
scattering, the reference light passing through the sample is
subjected to the phase change. The first reference light and the
second reference light passing through the sample are incident to a
second optical splitter 718.
[0061] The first reference light and the second reference light
split into the second path are reflected from a third reflector 716
and are incident to the second optical splitter 718. At this time,
in order to maximize the interference pattern, a variable
wavelength plate 717 may be disposed in the paths.
[0062] There is a phase difference between the first reference
light propagated to the first path passing through the sample S and
the second path that does not pass through the sample S. Therefore,
after the first reference light passes through the second optical
splitter 718, the interference phenomenon occurs. This interference
is interference at the frequency .omega..sub.ref1.
[0063] Likewise, there is a phase difference between the second
reference light propagated to the first path passing through the
sample S and the second reference light propagated to the second
reference light not passing through the sample S. Therefore, after
the second reference light passes through the second optical
splitter 718, the interference phenomenon occurs. This interference
is interference at the frequency .omega..sub.ref2.
[0064] The third dichroic reflector 719 passes through the light at
the frequency .omega..sub.ref1 and reflects the light at the
frequency .omega..sub.ref1. Therefore, the first detector 721
detects the signals due to the interference phenomenon at the
frequency .omega..sub.ref1 area and the second detector 722 detects
the signals due to the interference phenomenon at the frequency
.omega..sub.ref2 area reflected from a fourth reflector 720. The
signals detected in each of the first detector 721 and the second
detector 722 are input to an imaging unit 730. The imaging unit 730
images a difference between the signal detected in the first
detector 721 and the signal detected in the second detector 722 and
outputs it through the display (not shown) etc.
[0065] FIG. 8 is a diagram showing a configuration of an apparatus
for obtaining images using coherent anti-stokes Raman scattering
according to still another embodiment of the present invention. As
described with reference to FIG. 2, when the refractive index is
changed in the vicinity of the anti-stokes frequency upon
generating the coherent anti-stroke Raman scattering, the change in
birefringence is differently displayed according to directivity of
the refractive index. With the embodiment, the images of the sample
are obtained using the change in birefringence.
[0066] Referring to FIG. 8, an apparatus for obtaining images
according to the present invention includes a pump light source 801
and a stokes light source 802 that generate pump light and stokes
light irradiated on the sample to generate anti-stokes light having
anti-stokes frequency, a reference light source 810 that generates
reference light having higher or lower frequency by a predetermined
magnitude than the anti-stroke frequency, and as shown in FIG. 8,
an image obtaining unit that obtains images of the sample S using
the change in polarizing state of the reference light due to the
change in birefringence of the sample. As the pump light source 801
and the stokes light source 802, a pulse type laser light source is
used.
[0067] The pump light source 801 and the stokes light source 802
each generates the pump light at the frequency .omega..sub.p and
the stokes light at the frequency .omega..sub.s and the pump light
and the stokes light are reflected from a first dichroic reflector
814 and are irradiated on the sample S. In the sample, the
anti-stokes light is generated due to the coherent anti-stokes
Raman scattering. And, the pump light and the stokes light are
reflected from a second dichroic reflector 816 and emitted to the
outside. The first dichroic reflector 814 and the second dichroic
reflector 816 performs a function of filtering the pump light and
the stokes light by reflecting the pump light at the frequency
.omega..sub.p and the stokes light at the frequency .omega..sub.s
and passing through the reference light at the frequency
.omega..sub.ref.
[0068] The reference light source 810 generates the reference light
having the frequency .omega..sub.ref. The reference light becomes
light polarized in an optical axis direction owned by a first
polarizer 811 after passing through the first polarizer 811. The
polarized reference light passes through a variable wavelength
plate 812 and then reflected from the first reflector 813, which is
in turn irradiated on the sample S. At this time, the reference
light can be magnified by a microscope object lens 815. The
reference light passes through the sample S and the reflected from
the second reflector 817. And, the reference light is incident on
an optical axis in a different direction from the first polarizer
811, for example, a second polarizer 818 having the optical axis
having a difference by 90.degree.. A detector 819 detects the
reference light passing through the second polarizer 818 and an
imaging unit 830 analyzes polarizing property of the reference
light before passing through the sample S and polarizing property
of the reference light after passing through the sample S, measure
the birefringence, images it, and outputs it through the display
(not shown) etc.
[0069] FIG. 9 is a diagram showing a configuration of an apparatus
for obtaining images using coherent anti-stokes Raman scattering
according to still another embodiment of the present invention and
shows an embodiment further including an electromagnetic generating
unit 840 that applies electric field or magnetic field to the
sample S in the apparatus for obtaining images. As shown in FIG. 9,
if the electric field or the magnetic field is applied to the
sample S, the coherent anti-stroke Raman scattering effect can be
increased or reduced. Therefore, the change in birefringence due to
the coherent anti-stokes Raman scattering can be controlled.
[0070] With the present invention, the images of the sample can be
obtained by using the change in the refractive index or the change
in birefringence in the vicinity of the anti-stokes frequency owned
by the anti-stokes light without directly measuring the signal
strength of the anti-stokes light generated by the coherent
anti-stokes Raman scattering. Therefore, the present invention can
implement the apparatus for obtaining images using coherent
anti-stokes Raman scattering that is not affected by the
non-resonant background signal phenomenon and has strong resistance
against noise even if the signal is weak and has excellent
sensitivity and resolution since the change in the refractive index
has nothing to do with the signal strength
[0071] Although the preferred embodiment of the present invention
is described, it will be apparent to those skilled in the art that
various modifications and variations can be made in the present
invention without departing from the spirit or scope of the
inventions. Thus, it is intended that the present invention covers
the modifications and variations of this invention provided they
come within the scope of the appended claims and their
equivalents.
* * * * *